The present invention is directed to a humanized single chain antibody having a framework motif, preferably a motif containing no murine amino acids, that result in the humanized antibody having activity comparable to the corresponding murine antibody. Preferably, the antibody is a Tat antibody.
Human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2) are the etiologic agents of acquired immunodeficiency syndrome (AIDS) in humans (Barre-Sinoussi et aL., 1984). AIDS results from the depletion of CD4-positive T lymphocytes in HIV-infected individuals (Fauci et al., 1984).
HIV-1 infects T lymphocytes, monocytes/macrophage, dendritic cells and, in the central nervous system, microglia (Gartner et al., 1986; Koenig et al., 1986; Pope et al., 1994; Weissman et al., 1995). All of these cells express the CD4 glycoprotein, which serves as the receptor for HIV-1 and HIV-2 (Dalgleish et al., 1984; Klatzman et al., 1984; Maddon et al., 1986). Efficient entry of HIV-1 into target cells is dependent upon binding of the viral exterior envelope glycoprotein, gp120, to the CD4-amino-terminal domain (McDougal et al., 1986; Helseth et al., 1990). After virus binding, the HIV-1 envelope glycoproteins mediate the fusion of viral and host cell membranes to complete the entry process (Kowalski et al., 1987; Stein et al., 1987; Helseth et al., 1990). Membrane fusion directed by HIV-1 envelope glycoproteins expressed on the infected cell surface leads to fusion with uninfected CD4-positive cells, resulting in syncytia (Lifson et al., 1986; Sodroski et al., 1986). HIV-1 and HIV-2 contain numerous regulatory proteins including tat, rev, nef, vpu/vpx and vpr in addition to pol, gag and the envelope glycoproteins.
Tat, a 16 kD regulatory protein, is expressed early in the viral life cycle and is absolutely required for viral replications1,2. Tat acts as a potent transcriptional activator of viral gene expression through its binding to a RNA stem-loop structure called the transactivation response element (TAR) that is located 40 bp downstream from the site of initiation of transcription in the 5xe2x80x2 long terminal repeat (LTR). Tat functions primarily to stimulate transcription initiation and increase transcriptional elongation3,4,5. However, new evidence suggests that Tat may also be required for efficient HIV-1 reverse transcription6,7.
Apart from its role in viral replication, Tat protein also has an effect on cellular genes that may aid in the dissemination of virus infection. For example, Tat has been implicated in several immunosuppressive effects including increasing the expression of the potent immunosuppressive cytokine transforming growth factor xcex21 (TGF-xcex21)8, suppressing antigen-induced proliferation of T cells9 and decreasing the activity of an MHC class I gene promoter, thereby providing a mechanism whereby HIV-1-infected cells may be able to avoid immune surveillance and recognition of specific cytotoxic T lymphocytes10. Other cellular genes such as those involved in G1 checkpoint control, p53 and in cellular defense against oxidative stress, Mn-superoxide dismutase are also downregulated by Tat11,12.
Tat has additional functions in the pathogenesis of AIDS, in part because of its ability to be released from HIV-1-infected cells through a non-classical secretory pathway and to enter the nuclei of both infected and uninfected cells. Tat uptake not only enhances HIV-1 transcription in infected cells, it also affects a range of host cellular genes in both infected and uninfected cells. This includes activation of cellular genes such as tumor necrosis factor (TNF) xcex1 and xcex213,14,15 and IL-616,17, which in turn may activate HIV-1 gene expression and replication leading to further spread of HIV-118,19,20,21. Tat has also been shown to upregulate IL2 secretion in ctivated T cells22 and to recapitulate the phenotype of increased IL-2 secretion in response to costimulation with CD3 plus CD28 that is seen in HIV-1-infected primary T-cells that are stimulated via CD3 and CD28 receptors23. Extracellular Tat has been shown to activate uninfected quiescent T cells in vitro and in vivo, thereby causing uninfected cells to become highly permissive to productive HIV-1-infection24. In this way, Tat protein is unique among the HIV-1 proteins in not only being critical for ,iral transcriptional activation but also for its role in evolvinga self-perpetuating mechanism to actively generate cells permissive to productive and cytopathic infection24,25.
Consequently Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and on the immune system. It would be desirable to have more efficient means for disrupting Tat interactions. Disruption of Tat protein interaction with TAR RNA or the cellular factors that bind Tat protein, and of Tat protein release from HIV-1-infected cells, thus represents an important target for pharmacologically and genetically based therapeutic interventions to combat HIV-1 infection. While clinical results with the Tat antagonist Ro24-7429 showed no evidence of anti-viral activity26 despite prolonged inhibition of HIV-1 replication in vitro27, the results of a number of Tat directed in vitro gene therapy studies have been encouraging28,29,30,31,32,33,34, particularly when combined with pharmacologic inhibitors of NF-kB35,36.
A murine anti-tat sFv antibody, which is directed intracellularly against the proline-rich N-terminal activation domain of HIV-1 Tat and hence sometimes referred to as an intrabody, is a potent inhibitor of Tat-mediated LTR transactivation and HIV-1 infection36,37,38. However, murine antibodies can produce undesired immune responses which can reduce or totally abolish the effectiveness of the antibody. The immune response can also cause undesired side effects. In order to minimize evoking an immune response against the murine anti-tat sFv or transgene encoding it in a clinical setting39, CDR grafting experiments were performed to completely humanize the murine anti-tat sFv. Unfortunately, xe2x80x9chumanizingxe2x80x9d an antibody is not as efficient a process as sometimes presented. Compatible human framework regions must be chosen from heavy chain and light chain sequences of over 1000 human sequences each. However, the resulting antibody despite having the same variable region as the murine antibody frequently does not have the same effectiveness as the original murine antibody. Frequently the xe2x80x9chumanizedxe2x80x9d antibody will retain some xe2x80x9cmurinexe2x80x9d amino acid residues. It would be desirable to have a framework motif that produces an antibody having a protective efficiency comparable to the murine antibody.
We have now discovered a framework motif that produces a humanized antibody such as an anti-tat sFv intrabody that demonstrated a level of activity, e.g., anti-HIV-1 activity that was comparable to that of the parental murine sFv.
The preferred sequence was completely human, retaining none of the murine amino acids. The comparable human heavy chain and light chain are selected. One preferred framework motif is based upon the human VH gene K5B8 and VL gene TR1.6.
While the sequence is preferably completely humanized, some murine amino acid residues can be retained. The amino acid sequences encoded by these genes are aligned against the murine sequence to determine where the amino acids differ. One humanized antibody retains at least one of the murine amino acid residues at the FRM2/CDR2 border and the FRM3/CDR3 border of the heavy chain. In another embodiment, at least one murine amino acid within the FRM3 sequence of the light chain is also retained. Preferred positions are the first murine amino acid within FRM3 after the CDR2 border that differs from the human sequence and the last such amino acid within FRM3 before the CDR border. In yet another embodiment, at least three of the four murine positions described above are maintained. For example, all four of these murine amino acids are retained. In another embodiment the murine amino acid at the heavy chain CDR3/FR4 boundary is also maintained. This murine amino acid can be maintained with any of the above-described combination of murine and human amino acids. However, most preferably, the framework motif retains none of the murine sequences.
We have found, for example, that a humanized test antibody retaining no murine amino acids demonstrated a level of HIV-1 protective activity comparable to the parent murine sFv when transduced PBMC expressing the murine or humanized sFv antibodies were challenged with HIV isolates. In contrast, a humanized version retaining five of the murine amino acids and one retaining the murine amino acid at the heavy chain CDR3/FR4 boundary while demonstrating some level of protective activity do not demonstrate comparable protective activity.
These antibodies can be used in a variety of ways. For example, the DNA encoding such an antibody can be used to transform a cell which will then express the antibody intracellularly. For example, when the leader sequence is removed the antibody will not go to the endoplasmic reticulum (ER). In one embodiment the appropriate nuclear localization sequence is added, and the antibody can target a protein at a specific location intracellularly and prevent binding. In another embodiment, a protein such as the tat protein, can be targeted. in the cytoplasm by deleting the leader sequence without adding a nuclear localization sequence.
For example, the antibody sFvtat1Ck, a murine anti-tat sFv intrabody, directed against the proline rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication (EMBO J. 14:1542, 1995). Stably transfected CD4 SupT1 cells expressing this intrabody were resistant to HIV-1 infection at high m.o.i. with both the laboratory isolate Hxc3x97B2 and six syncytium inducing (SI)-primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis upon addition of Phorbol 12-myristate 13 acetate (PMA), which is equal to 12-0-Tetradecanoylphorbyl xcex2-acetate (TPA), or Tumor necrosis factor-alpha (TNFxcex1), showed decreased production of HIV-1 in the presence of sFvtat1 Ck. In transduced CD4+-selected, CD8+-depleted and total PMBCs, the sFvtat1Ck expressing cells showed marked inhibition of HIV-1 replication. A humanized antibody prepared,by substituting compatible human framework regions chosen from a large database of human VH and VL sequences on the basis of high overall framework matching, similar CDR length and minimal mismatching of canonical and VH/VL contact residues altered as taught, sFvhutnat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI-primary isolates. However, as mentioned above, the other humanized antibody did not display such activity.
These antibodies can also be used extracellularly to target Tat. The antibodies can also be used to bind to Tat and when combined with a detectable moiety used to measure levels of Tat. The Tat levels can be used diagnostically and/or prognostically.